Icreasing envidence points to a central role of extracellular matrix vesicles in biological calcification. The objective of the proposed research is to elucidate the mechanism whereby matrix vesicles initiate and regulate endochondral calcification. Emphasis will be focused on further biochemical characterization of the matrix vesicles re. enzymes, calcium- and phosphate-binding and transport proteins, mineral forms, and membrane components, and on further elucidation of their mineral metabolism (45Ca and 32Pi transport and deposition), and their formation and degradation (esp. lipid biosynthesis and degradation). Special effort will be made to elucidate the roles of alkaline phosphatase, magnesium, nucleotides and phosphorylated metabolites, and the acidic phospholipid-Ca-PI complexes. Our experimental system will continue to be the epiphyseal plate of the long bones of rapidly-growing broiler chickens. However our newly-developed non-enzymatic method for isolating matrix vesicles involves numerous centrifugation steps which require preparative ultracentrifugation. Further characterization of these actively calcifying structures depends on our ability to rapidly prepare them and to analyze their chemical components and metabolic activities. The techniques we plan to use include conventional chemical analyses, various chromatographic, electrophoretic, spectroscopic and enzymatic methods. A particularly powerful tool for obtaining rapid and accurate analysis of chemical constituents is the newly-developed system of high-performance liquid chromatography (HPLC). The purpose of this grant supplement is to request reinstatement of funds for an HPLC system and for a preparative ultracentrifuge. These items were requested in our original application, but were not approved, primarily I believe, because we fail to provide adequate documentation. In this supplemental request we hope to outline and justify our need for both of these instruments and a small refrigerated clinical centrifuge.